[Effect of Golgi α-mannosidase 2 (GM2) gene knockdown on adhesion abilities of human gastric carcinoma cell line BGC-823 and its mechanism].

Objective To investigate the effect of Golgi α-mannosidase II (GM2) gene knockdown on adhesion abilities of BGC-823 human gastric carcinoma cells. Methods Three plasmid vectors expressing GM2 shRNAs and a negative control plasmid vector were designed, constructed and then transfected into BGC-823 cells by LipofectamineTM 2000. After transfection, the mRNA and protein levels of GM2 in BGC-823 cells were detected by real-time quantitative PCR (qRT-PCR) and Western blotting to evaluate the transfection efficacy. The best plasmid for GM2 gene knockdown was selected and stably transfected into BGC-823 cells. Adhesion abilities of BGC-823 cells after GM2 gene silencing were observed by cell-cell, cell-matrix and cell-endothelial cell adhesion assays. At the same time, the expressions of E-cadherin, P-selectin, CD44v6 and intercellular adhesion molecule-1 (ICAM-1) proteins were detected by Western blotting after GM2 gene knockdown. Results The expression of GM2 was effectively knockdown in GM2-shRNA-2-transfected BGC-823 cells. Compared with the blank control group and the negative control group, the intercellular adhesion ability of the GM2-shRNA-2-transfected cells increased significantly, while their cell-matrix and cell-endothelium adhesion abilities markedly decreased. In GM2-shRNA-2 transfection group, E-cadherin expression was significantly elevated and the P-selectin expression was significantly reduced, while the expression levels of CD44v6 and ICAM-1 were not obviously changed. Conclusion After GM2 gene knockdown, the intercellular adhesion ability of gastric carcinoma BGC-823 cells is enhanced, while the adhesion abilities with the extracellular matrix and endothelial cells are weakened. The changes might be related to the up-regulated expression of E-cadherin and the down-regulation of P-selectin.

MAN3 gene regulates cadmium tolerance through the glutathione-dependent pathway in Arabidopsis thaliana.

Pollution of soil by the heavy metal cadmium (Cd) is a global environmental problem. The glutathione (GSH)-dependent phytochelatin (PC) synthesis pathway is one of the most important mechanisms contributing to Cd accumulation and tolerance. However, the regulation of this pathway is poorly understood. Here, we identified an Arabidopsis thaliana cadmium-tolerant dominant mutant xcd1-D (XVE system-induced cadmium-tolerance 1) and cloned XCD1 gene (previously called MAN3), which encodes an endo-ß-mannanase. Overexpression of MAN3 led to enhanced Cd accumulation and tolerance, whereas loss-of-function of MAN3 resulted in decreased Cd accumulation and tolerance. In the presence of estradiol, enhanced Cd accumulation and tolerance in xcd1-D was associated with GSH-dependent, Cd-activated synthesis of PCs, which was correlated with coordinated activation of gene expression. Cd stress-induced expression of MAN3 and the consequently increased mannanase activity, led to increased mannose content in cell walls. Moreover, mannose treatment not only rescued the Cd-sensitive phenotype of the xcd1-2 mutant, but also improved the Cd tolerance of wild-type plants. Significantly, this mannose-mediated Cd accumulation and tolerance is dependent on GSH-dependent PC concentrations via coordinated control of expression of genes involved in PC synthesis. Our results suggest that MAN3 regulates the GSH-dependent PC synthesis pathway that contributes to Cd accumulation and tolerance in A. thaliana by coordinated control of gene expression.

ERManI is a target of miR-125b and promotes transformation phenotypes in hepatocellular carcinoma (HCC).

The MAN1B1 gene product, designated ER alpha-1, 2-mannosidase (ERManI), is an enzyme localized in the Golgi complex of mammalian cells. By functioning as a "gate keeper" to prevent the inappropriate secretion of misfolded glycoproteins, it plays a critical role in maintaining protein homeostasis in the mammalian secretory pathway. In the present study, we identified that a conserved motif within the 3'UTR of ERManI is a target of miR-125b, a microRNA frequently down-regulated in numerous types of cancers, including hepatocellular carcinoma (HCC). As predicted, the expression of ERManI is significantly elevated in HCC, as measured by immunohistochemistry in a liver spectrum tissue microarray. Additional analyses using several hepatoma cell lines demonstrated that the elevated ERManI inversely correlates with a diminished intracellular concentration of miR-125b. Moreover, functional studies indicated that RNAi-mediated knock-down of endogenous ERManI was sufficient to inhibit proliferation, migration, and invasion of hepatoma cells. These phenotypical changes occurred in the absence of alterations in global glycoprotein secretion or ER-stress status. Together, these results revealed a novel post-transcriptional regulatory mechanism for ERManI and implied that this molecule contributes to the regulation of carcinogenesis in HCC independent of its function in glycoprotein quality control.

Endo-beta-mannosidase, a plant enzyme acting on N-glycan: purification, molecular cloning, and characterization.

Endo-beta-mannosidase is a novel endoglycosidase that hydrolyzes the Manbeta1-4GlcNAc linkage in the trimannosyl core structure of N-glycans. This enzyme was partially purified and characterized in a previous report (Sasaki, A., Yamagishi, M., Mega, T., Norioka, S., Natsuka, S., and Hase, S. (1999) J. Biochem. 125, 363-367). Here we report the purification and molecular cloning of endo-beta-mannosidase. The enzyme purified from lily flowers gave a single band on native-PAGE and three bands on SDS-PAGE with molecular masses of 42, 31, and 28 kDa. Amino acid sequence information from these three polypeptides allowed the cloning of a homologous gene, AtEBM, from Arabidopsis thaliana. AtEBM was engineered for expression in Escherichia coli, and the recombinant protein comprised a single polypeptide chain with a molecular mass of 112 kDa corresponding to the sum of molecular masses of three polypeptides of the lily enzyme. The recombinant protein hydrolyzed pyridylamino derivatives (PA) of Manalpha1-6Manbeta1-4Glc-NAcbeta1-4GlcNAc into Manalpha1-6Man and GlcNAcbeta1-4Glc-NAc-PA, showing that AtEBM is an endo-beta-mannosidase. AtEBM hydrolyzed Man(n)Manalpha1-6Manbeta1-4GlcNAcbeta1-4GlcNAc-PA (n = 0-2) but not PA-sugar chains containing Manalpha1-3Manbeta or Xylosebeta1-2Manbeta as for the lily endo-beta-mannosidase. AtEBM belonged to the clan GH-A of glycosyl hydrolases. Site-directed mutagenesis experiments revealed that two glutamic acid residues (Glu-464 and Glu-549) conserved in this clan were critical for enzyme activity. The amino acid sequence of AtEBM has distinct differences from those of the bacterial, fungal, and animal exo-type beta-mannosidases. Indeed, AtEBM-like genes are only found in plants, indicating that endo-beta-mannosidase is a plant-specific enzyme. The role of this enzyme in the processing and/or degradation of N-glycan will be discussed.

Elucidation of the molecular logic by which misfolded alpha 1-antitrypsin is preferentially selected for degradation.

The exocytic pathway provides a physical route through which newly synthesized secretory and membrane proteins are deployed to the eukaryote cell surface. For newly synthesized alpha1-antitrypsin (AAT), the modification of its asparagine-linked oligosaccharides by a slow-acting mannosidase partitions the misfolded monomer into the proteasomal degradation pathway. Herein, we asked whether, and how, modification by endoplasmic reticulum mannosidase I (ERManI) contributes to the preferential selection of the misfolded AAT monomer for proteasomal degradation. Transiently expressed mutant and WT AAT variants underwent rapid destabilization in response to an artificially elevated ERManI concentration in the murine hepatoma cell line, Hepa1a. Based on the mannosidase- and lactacystin-sensitive properties of intracellular turnover, a stochastic model is proposed in which the delayed onset of the glycan modification, relative to the duration of nonnative protein structure, coordinates the preferential degradation of the misfolded monomer and spares the native molecule from destruction. Newly synthesized endogenous transferrin underwent degradation in response to an elevated concentration of ERManI, whereas the nonglycosylated secretory glycoprotein albumin was not affected. Taken together, these findings indicate that efficient conformational maturation might function as the initial quality control standard for a broad population of glycoproteins.

The role of N-glycans in spermatogenesis.

Many proteins, in particular those in the plasma membranes, are glycosylated with carbohydrates, which are grouped into O-glycans and N-glycans. O-glycans are synthesized step by step by glycosyltransferases, whereas N-glycans are synthesized by en-bloc transfer of the so-called high-mannose-type oligosaccharide from lipid-linked precursor to polypeptide. The high-mannose-type N-glycans are then modified by processing alpha-mannosidases. Alpha-mannosidase IIx (MX) was identified as the gene product of processing alpha-mannosidase II (MII)-related gene. MX apparently plays subsidiary role for MII in many cell types, as N-glycan patterns of MX null mouse tissues are not altered significantly. Surprisingly MX null male mice are infertile due to a failure of spermatogenesis. This review provides a brief overview of the in vivo role of N-glycans which are revealed by the gene knockout mouse approach, and introduce our studies on the MX gene knockout mouse. The MX gene knockout experiments unveiled a novel function of a specific N-glycan, which is N-acetylglucosamine-terminated and has a fucosylated triantennary structure, in the adhesion between germ cells and Sertoli cells. The study of MX is a good example of how the in vivo roles of an apparently redundant gene product are determined by the gene knockout approach.

Molecular cloning, chromosomal mapping and tissue-specific expression of a novel human alpha1,2-mannosidase gene involved in N-glycan maturation.

Class I alpha1,2-mannosidases play an essential role in the elaboration of complex and hybrid N -glycans in mammalian cells. Using degenerate primers based on amino acid sequences conserved in all members of this enzyme family for RT-PCR, two distinct PCR products were obtained from placenta and lymphocyte cDNAs. One of these was related to the previously cloned human and murine alpha1, 2-mannosidase IA whereas the other was very similar to murine alpha1, 2-mannosidase IB. Northern blot analysis of human tissues with these two alpha1,2-mannosidase probes revealed very different patterns of tissue-specific expression. Similar tissue-specific expression of alpha1,2-mannosidase IA and IB was also observed on Northern blots of adult mouse tissues. A human placenta cDNA library was screened and PCR of brain, placenta, and lymphocyte cDNAs was performed in order to isolate the human alpha1,2-mannosidase IB cDNA. This cDNA encodes a type II membrane protein of 73 kDa that is 94% identical in amino acid sequence to the murine alpha1,2-mannosidase IB (Herscovics et al., 1994, J. Biol. Chem., 269, 9864-9871). A truncated soluble form of the human alpha1,2-mannosidase IB lacking its N -terminal transmembrane domain was expressed as a secreted protein in Pichia pastoris . The recombinant enzyme was incubated with [3H]Man9GlcNAc and [3H]Man8GlcNAc (isomer B), and high performance liquid chromatography analysis of the products showed that [3H]Man9GlcNAc was readily converted to [3H]Man6GlcNAc and much more slowly to [3H]Man5GlcNAc, whereas [3H]Man8GlcNAc was rapidly trimmed to [3H]Man5GlcNAc. The human alpha1,2-mannosidase IB gene was isolated from a P1 human genomic library and shown to be at least 60 kb in size and to contain at least 13 exons. The gene was localized by fluorescence in situ hybridization to human chromosome 1p13, a region that undergoes many aberrations in various types of human cancers. These results show that there are at least two Class I alpha1,2-mannosidases in the human and murine genomes with very distinct transcriptional regulation in different tissues.

The conserved noncatalytic 40-residue sequence in cellulases and hemicellulases from anaerobic fungi functions as a protein docking domain.

Two cDNAs, designated xynA and manA, encoding xylanase A (XYLA) and mannanase A (MANA), respectively, were isolated from a cDNA library derived from mRNA extracted from the anaerobic fungus, Piromyces. XYLA and MANA displayed properties typical of endo-beta 1,4-xylanases and mannanases, respectively. Neither enzyme hydrolyzed cellulosic substrates. The nucleotide sequences of xynA and manA revealed open reading frames of 1875 and 1818 base pairs, respectively, coding for proteins of M(r) 68,049 (XYLA) and 68,055 (MANA). The deduced primary structure of MANA revealed a 458-amino acid sequence that exhibited identity with Bacillus and Pseudomonas fluorescens subsp. cellulosa mannanases belonging to glycosyl hydrolase Family 26. A 40-residue reiterated sequence, which was homologous to duplicated noncatalytic domains previously observed in Neocallimastix patriciarum xylanase A and endoglucanase B, was located at the C terminus of MANA. XYLA contained two regions that exhibited sequence identity with the catalytic domains of glycosyl hydrolase Family 11 xylanases and were separated by a duplicated 40-residue sequence that exhibited strong homology to the C terminus of MANA. Analysis of truncated derivatives of MANA confirmed that the N-terminal 458-residue sequence constituted the catalytic domain, while the C-terminal domain was not essential for the retention of catalytic activity. Similar deletion analysis of XYLA showed that the C-terminal catalytic domain homologue exhibited catalytic activity, but the corresponding putative N-terminal catalytic domain did not function as a xylanase. Fusion of the reiterated noncatalytic 40-residue sequence conserved in XYLA and MANA to glutathione S-transferase, generated a hybrid protein that did not associate with cellulose, but bound to 97- and 116-kDa polypeptides that are components of the multienzyme cellulase-hemicellulase complexes of Piromyces and Neocallimastix patriciarum, respectively. The role of this domain in the assembly of the enzyme complex is discussed.

Effect of inhibitors of N-linked oligosaccharide processing on the cell surface expression of a melanoma integrin.

The role of trimming and processing of N-linked oligosaccharides on the cell surface expression of the melanoma vitronectin receptor, a member of the integrin family of cell adhesion receptors, was examined by using specific glucosidase and mannosidase inhibitors. Inhibition of glucosidases I and II by castanospermine or N-methyldeoxynojirimycin delayed the vitronectin receptor alpha/beta chain heterodimer assembly and alpha chain cleavage and resulted in a decrease in the level of expression cell surface receptor. Conversely, the vitronectin receptor synthesized in the presence of the mannosidase I and II inhibitors, 1-deoxymannojirimycin and swainsonine, was transported normally to the cell surface with its alpha chain N-linked oligosaccharides in an endoglycosidase H-sensitive form. In the presence of swainsonine, time course studies of the cell surface replacement of control, endoglycosidase H-resistant receptor with an endoglycosidase H-sensitive form demonstrated a vitronectin receptor half-life of approximately 15-16 h. These studies provide evidence that the rates of assembly, proteolytic cleavage, and cell surface expression of the melanoma vitronectin receptor are dependent on the initial trimming of glucosyl residues from the alpha chain N-linked oligosaccharides.